“Gene therapy” is the use of DNA as an agent to treat disease. It derives its name from the idea that DNA can be used to supplement or alter genes within a patient's cells as a therapy to treat disease. The most common form of gene therapy involves using DNA that encodes a functional, therapeutic gene to replace a mutated, non-functional gene.
Although early clinical failures led many to dismiss gene therapy as over-hyped, clinical successes have now bolstered new optimism in the promise of gene therapy. These include successful treatment of patients with the retinal disease Leber's congenital amaurosis, X-linked severe combined immunodeficiency (SCID), adenosine deaminase SCID (ADA-SCID), adrenoleukodystrophy, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), multiple myeloma and Parkinson's disease. These recent clinical successes have led to a renewed interest in gene therapy, with several articles in scientific and popular publications calling for continued investment in the field.
RNA is used in antisense and siRNA based therapies, but to date mRNA has not been used per se for gene therapy, even though the use of mRNA versus DNA in gene therapy offers potential advantages. For example, the protein encoded by the mRNA will be expressed in all cells, so selection of a promoter is not a problem. No insertional mutagenesis can occur, increasing the safety of the method, and the transient nature of expression is advantageous for many applications. The gene of interest can be easily expressed in dividing or non-dividing cells, as opposed to the limitations of DNA.
However, there are considerable technical difficulties to overcome before mRNA can be successfully used in various therapeutic methods.
For example, transfecting mRNA using lipids, electroporation, and other methods results in an inflammatory immune response mediated by Toll-like receptors recognizing the added RNA as foreign. This recognition leads to interferons being secreted, and if mRNA is attempted for repeated transfection, then ultimately cell death occurs via apoptosis.
A recent breakthrough allows the innate immune response to be avoided, thus providing a way of overcoming this first hurdle. The strategy incorporates modified nucleotides that cannot bind to toll-like receptors into the RNA, thus preventing the inflammatory immune response (e.g., U.S. Pat. No. 8,278,036, US20100047261, US20120322864). Thus, at least one challenge has been overcome in the challenges for implementing RNA-based therapeutic techniques.
Another difficulty has been the production of a complete and active mRNA via in vitro transcription. Further, the resulting mRNA must have all of the features needed for initiation and translation, and be able to effectively compete against endogenous mRNAs. Thus, the complete mRNA in the current art needs a 5′ cap or cap analogue, 5′ UTR, ORF, 3′ UTR, and polyadenylation tail to mimic the standard mRNA molecule produced by eukaryotic cells. In some cases, a 5′ cap is omitted and an IRES sequence utilized, but this is much more inefficient and reduces the half-life of the linear RNA molecule with no protection of the 5′ terminus of RNA. Similarly, a polyadenylation tail can be omitted, but with reduced translation efficiency and half-life of the linear mRNA molecule.
Perhaps the biggest impediment, however, is the difficulty in handling mRNA. RNA has two adjacent pendant hydroxyls on the pentose ring of the terminal nucleotide, making it very susceptible to nucleophilic attack by bases or by ever-present RNAses in water and on most surfaces. RNAse-free reagents are used for the production of mRNA and its resultant storage, but even with such techniques, the extreme sensitivity to degradation presents considerably difficulty in implementing any RNA based technique. Yet another impediment is the short half-life of mRNA once inside the cell. Messenger RNA only affords transient expression inside cells, generally on the order of 6-12 hours.
It is well appreciated in the literature that circular RNA molecules have much longer half-lives than their linear counterparts, being naturally resistant to any exonuclease activity or nucleophilic attack. Thus, the use of circular RNA can solve both of these degradation issues. In fact, the half-life of circular RNA in vivo was estimated to be greater than 40 hours in Xenopus embryos. In the same system, linear mRNAs had a half-life of 6-8 hours. Even in E. coli, a circular RNA being actively translated was 4-6 times more stable than its linear counterpart due to resistance to RNase E activity.
It is also known that a Shine-Dalgarno sequence is necessary in prokaryotes for ribosome recruitment and can mediate recruit of ribosomes to any RNA molecule, whether linear or circular. However, circular RNA was originally thought to be unable to bind to eukaryotic ribosomes. Fortunately, Chen (1995) demonstrated that circular mRNA can bind eukaryotic ribosomes with the presence of an internal ribosome entry site (IRES).
Chen utilized a picornavirus IRES sequence for this purpose and demonstrated translation in an in vitro rabbit reticulocyte system. The primary goal of their strategy focused on the application of developing polymeric proteins through continuous translation around the circular RNA molecule. In order for this to occur, they eliminated the stop codon so that the ribosome would never be signaled to fall off the RNA molecule. In such constructs, only the IRES site and the coding sequence was present in the mRNA molecule, and other signals such as UTRs, polyA tracts, terminations sites and the like were missing.
In summary, for eukaryotes, a circular mRNA expression system has only been demonstrated in vitro in rabbit reticulocytes, a system that otherwise biases any level of background translation, even on a linear template without cap or IRES sequences. There was no data presented for the ability of a circular mRNA to translate in vivo inside a eukaryotic cell, and results in prokaryotes were disappointing. For application to an in vivo translation system inside the cell, more modifications are needed to circular mRNA in order to allow for its successful competition with native cellular mRNAs for translation initiation factors.
The Sarnow and Chen patent (U.S. Pat. No. 5,766,903) claims the insertion of an IRES into a circular RNA with a gene of interest. However, this patent fails to describe the necessity of other regulatory elements in the circular RNA molecule for in vivo translation. Indeed, there is no data demonstrating successful intracellular translation of circular mRNA in the patent or publication literature. There is no discussion of the insertion of a polyadenylation sequence, or a 3′ UTR to function in synergy with the IRES element. Furthermore, novel IRES elements with improved translation in circular mRNA were not proposed.
Furthermore, there were almost no follow-up reports in the literature demonstrating the utility of circular mRNA, in vitro or in vivo. In one recent work, it was shown in a rabbit reticulocyte system in vitro that a circular mRNA template with the SP-A1 IRES could direct translation (Wang 2009). However, the translation efficiency of circular RNA in vitro was 15% that of an uncapped linear RNA with IRES. In the same experiment, a capped linear RNA had an activity that was 131% that of uncapped linear RNA, emphasizing how the rabbit reticulocyte system tends to bias uncapped transcripts toward levels of translation that are super-physiologic.
A variety of additional patents concern circular mRNA. However, these patents fail to provide evidence of actual in vivo translation of the circular mRNA molecule. Examples of prior art include U.S. Pat. No. 5,766,903, U.S. Pat. No. 6,210,931, U.S. Pat. No. 5,773,244 U.S. Pat. No. 5,580,859, US20100137407, U.S. Pat. No. 5,625,047, U.S. Pat. No. 5,712,128 US20110119782. Therefore, although possibly recognizing the potential of using circular mRNA for in vivo expression in eukaryotes, such applications were not in fact enabled.
Thus, what is needed in the art are methods of making and using circular mRNA where such molecules have been fully enabled and shown to work in in vivo or ex vivo eukaryotic systems.